Methods and systems for synthesis of a d-aminoluciferin precursor and related compounds

ABSTRACT

Methods and systems to generate 6-amino-6-deoxy- D -luciferin precursor, 2-cyano-6-aminobenzothiazole and related compounds and derivatives

CROSS REFERENCE TO RELATED APPLICATIONS

This application is related and claims priority to U.S. ProvisionalApplication No. 61/308,317 entitled “Alternate Syntheses of Precursor toD-Aminoluciferin” filed on Feb. 26, 2010, docket IL-12088 to U.S.Provisional Application No. 61/331,072 entitled “Bioluminescent ProteaseProbe For The Detection Of Disease” filed on, May 4, 2010 with docketnumber IL-12087, and to U.S. Provisional Application No. 61/331,094entitled “Carboxylate-Modified Luciferin, Amino Acid/Peptide Probes ForBioluminescent Protease Assays” filed on, May 4, 2010 with docket numberIL-11088, each of which is incorporated herein by reference in itsentirety.

STATEMENT OF GOVERNMENT INTEREST

The United States Government has rights in this invention pursuant toContract No. DE-AC52-07NA27344 between the United States Department ofEnergy and Lawrence Livermore National Security, LLC for the operationof Lawrence Livermore National Laboratory

FIELD

The present disclosure relates to bioluminescence and in particular tomethods and systems for the synthesis of D-aminoluciferin precursor andrelated compounds.

BACKGROUND

Bioluminescence provides a useful read out for the detection ofreactions in a background of optically complex materials such as cellsand tissues and the signal-to-noise ratio can be very high.

Luciferases are enzymes that generate visible light through theoxidation of a specific substrate in the presence of oxygen and usuallya source of energy (such as Mg2⁺ and ATP). The reaction between fireflyluciferase and the substrate luciferin, yields oxyluciferin, carbondioxide and visible light with a maximum around 560 nm and with otherwavelengths of light output identifiable by a skilled person.

For these reasons, bioluminescence and in particular luciferin andrelated compounds are routinely used for in vivo imaging applicationsand for additional techniques for in vitro and/or in vivo detection oftargets and/or reactions.

SUMMARY

Provided herein are methods and systems to synthesize a precursor ofD-aminoluciferin and related compounds and methods that in severalembodiments allow to minimize mixture of products evident in the earlysteps and/or to conjugate D-aminoluciferin to amino acids and peptidesequences to generate probes suitable for bioluminescence assays.

According to a first aspect a method and system to provide 2-cyano6-amino-benzothiazole from a monofunctional benzothiazole is described.The method comprises providing a monofunctional benzothiazole attachingin position C2 a functional group of formula (I) (C(═X₁)NH₂) wherein X₁is O or S, and converting the functional group of formula (I) to acyanide group, through elimination of H₂X₁ from the monofunctionalbenzothiazole. In the method the monofunctional benzothiazole eithercomprises an amino group in position C6 or is modified to comprise anamino group in position C6. The system comprises one or moremonofunctional benzothiazoles attaching in position C2 a functionalgroup of formula (I), together suitable reagents for simultaneouscombined or sequential use in the method to provide2-cyano-6-aminobenzothiazole herein described

According to a second aspect a method and system to provide2-cyano-6-aminobenzothiazole from a monofunctional benzothiazole isdescribed. The method comprises providing a monofunctional benzothiazoleattaching in position C2 a functional group of formula (II) (C(═X₁)X₂R)wherein R is an alkyl group, or a halogen atom; and X₁ and X₂ areindependently O or S. The method also comprises converting thefunctional group of formula (II) into an amide of formula (I)(C(═X₁)NH₂) and converting the functional group of formula (I) to acyanide group through elimination of a H₂X₁ from the monofunctionalbenzothiazole. In the method the monofunctional benzothiazole eithercomprises an amino group in position C6 or is modified to comprise anamino group in position C6. The system comprises at least two of one ormore monofunctional benzothiazole attaching in position C2 a functionalgroup of formula (I), and/or one or more monofunctional benzothiazoleattaching in position C2 a functional group of formula (II) togethersuitable reagents for simultaneous combined or sequential use in themethod to provide 2-cyano-6-aminobenzothiazole herein described.

According to a third aspect, a method and system to provide an aminoacid labeled with 6-amino-6-deoxy-D-luciferin and amino acid obtainablethereby, are described. The method comprises conjugating the amino acidwith a 2-cyano-6-aminobenzothiazole to provide an amino acid-conjugated2-cyano-6-aminobenzothiazole or 2-cyano-6-aminobenzothiazole-[aminoacid, the conjugating performed to allow formation of a peptide bond, acarbamate bond or a urea/thiourea bond between the amino group of6-amino-6-deoxy-D-luciferin and a carboxylic group of the amino acid.The method further comprises reacting the2-cyano-6-aminobenzothiazole-[amino acid] with a D-cysteine to providean amino acid-conjugated 6-amino-6-deoxy-D-luciferin. In the method, theamino acid is an amino acid having a side chain. The system comprises atleast two of one or more amino acids with each amino acid having a sidechain an amino acid-conjugated 2-cyano-6-aminobenzothiazole-[amino acid]and an amino acid-conjugated 2-cyano-6-aminobenzothiazole-[amino acid]for simultaneous combined or sequential use in the method to provide anamino acid labeled with 6-amino-6-deoxy-D-luciferin herein described.

According to a fourth aspect an intermediate in the synthesis of anamino acid labeled with 6-amino-6-deoxy-D-luciferin is described. Theintermediate is a 2-cyano-6-aminobenzothiazole of formula (III)

wherein R1 is an amino acid attached to the remainder of the compound offormula (III) through a peptide bond, a carbamate bond or aurea/thiourea bond.

According to a fifth aspect, a method and system to provide a peptidelabeled with 6-amino-6-deoxy-D-luciferin is described. The methodcomprises conjugating the peptide with an amino acid-conjugated2-cyano-6-aminobenzothiazole to provide a peptide-conjugated2-cyano-6-aminobenzothiazole, the conjugating performed to allowformation of a peptide bond between the amino group of the aminoacid-conjugated 2-cyano-6-aminobenzothiazole and a carboxylic acid groupof the peptide. The method can further comprise reacting thepeptide-conjugated 2-cyano-6-aminobenzothiazole with D-cysteine toprovide a peptide-conjugated 6-amino-6-deoxy-D-luciferin. The systemcomprises one or more peptides, amino acids,2-cyano-6-aminobenzothiazole, 2-cyano-6-amino-aminoacid-benzothiazolesand/or 2-cyano-6-amino-peptide-benzothiazoles for simultaneous combinedor sequential use in the method to provide a peptide labeled with6-amino-6-deoxy-D-luciferin herein described.

According to a sixth aspect an intermediate in the synthesis of apeptide labeled with 6-amino-6-deoxy-D-luciferin is described. Theintermediate is a 2-cyano-6-aminobenzothiazole of formula (IV)

wherein R1 is a single amino acid and R2 is a peptide attached to theremainder of the compound of formula (IV) through a peptide bond acarbamate bond or a urea/thiourea bond.

According to a seventh aspect, a labeled peptide is described. Thelabeled peptide is obtainable by the method to provide a peptideconjugated to 6-amino-6-deoxy-D-luciferin herein described.

According to an eighth aspect a carboxylate-modified luciferin aminoacid/peptide probe, is described and related method for synthesis and/oruse of said carboxylate modified probe. The carboxylate-modifiedD-luciferin, amino acid/peptide probe comprises a D-luciferin moleculeconjugated to an amino acid or peptide wherein conjugation between theD-luciferin and the amino acid or peptide is performed with peptide bondbetween the amino-terminus of the amino acid or peptide and a carboxylgroup of the said D-luciferin.

The methods and systems described allow in several embodimentsfacilitating the generation of amounts of pure6-amino-6-deoxy-D-luciferin (D-aminoluciferin) precursor.

The methods and systems described allow in several embodimentsfacilitating the coupling of 6-amino-6-deoxy-D-luciferin(D-aminoluciferin) precursor to peptide sequences and/or to single aminoacids in solution phase.

The methods and systems described allow in several embodimentsgeneration of pure 6-amino-6-deoxy-D-luciferin (D-aminoluciferin)precursor and/or subsequent coupling to peptide sequences and/or tosingle amino acids in a solution phase.

The methods and systems described herein can be used in connection withapplications wherein production of D-aminoluciferin and/or related usesand/or derivatives is desired, including but not limited to medicalapplication, biological analysis and diagnostics including but notlimited to clinical applications.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description and theexamples, serve to explain the principles and implementations of thedisclosure.

FIG. 1 shows a schematic representation of a bioluminescent assay basedon release of D-aminoluciferin from labeled compounds according to anembodiment herein described.

FIG. 2 shows a schematic representation of a method to provide6-amino-2-cyanobenzothiazole from a monofunctional benzothiazoleaccording to an embodiment herein described. In particular, theschematic of FIG. 2 illustrates a synthetic route of a 4 step process togenerate D-aminoluciferin precursor, 2-cyano-6-aminobenzothiazolebeginning with a monofunctional benzothiazole(6-nitrobenzothiazole).

FIG. 3 shows a schematic representation of the four steps depicted inFIG. 2 according to an embodiment herein described. In particular, theschematic of FIG. 3 shows a specific route where a carboxyethylfunctional group is introduced in the position C2 of6-nitrobenzothiazole and then converted to a carboxamide group and on toa cyano group. The nitro group in position C6 of the resulting2-cyano-6-nitrobenzothiazole is then reduced to an amino group.

FIG. 4 shows nuclear magnetic resonance spectra for compounds of methodsand systems of the disclosure, according to an embodiment hereindescribed. FIG. 4A shows a nuclear magnetic resonance (¹H-NMR) spectraof ethyl 6-nitrobenzothiazole-2-carboxylate 2 obtained on a Bruker 500MHz machine in d₆-DMSO. FIG. 4B shows nuclear magnetic resonance(¹³C-NMR) spectra of ethyl 6-nitrobenzothiazole-2-carboxylate 2 obtainedon a Bruker 500 MHz machine in d₆-DMSO. FIG. 4C shows a nuclear magneticresonance (¹H-NMR) spectra of 6-nitrobenzothiazole-2-carboxamide 3obtained on a Bruker 500 MHz machine in d₆-DMSO. FIG. 4D shows Nuclearmagnetic resonance (¹³C-NMR) spectra of6-nitrobenzothiazole-2-carboxamide 3 obtained on a Bruker 500 MHzmachine in d₆-DMSO. FIG. 4E shows a nuclear magnetic resonance (¹H-NMR)spectra of 2-cyano-6-nitrobenzothiazole 4 obtained on a Bruker 500 MHzmachine in d₆-DMSO. FIG. 4F shows a nuclear magnetic resonance (¹³C-NMR)spectra of 2-cyano-6-nitrobenzothiazole 4 obtained on a Bruker 500 MHzmachine in d₆-DMSO. FIG. 4G shows a nuclear magnetic resonance (¹H-NMR)spectra of 2-cyano-6-aminobenzothiazole 5 obtained on a Bruker 500 MHzmachine in d₆-DMSO. FIG. 4H shows a nuclear magnetic resonance (¹³C-NMR)spectra of 2-cyano-6-aminobenzothiazole 5 obtained on a Bruker 500 MHzmachine in d₆-DMSO. FIG. 4I shows Mass spectra were acquired on aMicromass Quattro Micro API mass spectrometer operating in positive ionmode. The samples were dissolved in MeCN/H₂O (1:1), 0.1% formic acid formass spectrometry analysis.

FIG. 5 shows schematic representation of a method to provide2-cyano-6-aminobenzothiazole from a monofunctional benzothiazoleaccording to an embodiment herein described. In particular, theillustration of FIG. 5 shows a general scheme for syntheses of2-cyano-6-aminobenzothizaole, starting with a ethylbenzothiazole-2-carboxylate according to an embodiment herein described.

FIG. 6 shows a schematic representation of the four steps depicted inFIG. 5 according to an embodiment herein described. In particular, inthe illustration of FIG. 6A, the ethyl benzothiazole-2-carboxylate isfirst nitrated to provide a ethyl 6-nitrobenzothiazole-2-carboxylate;the ester group in position C2 of the resulting ethyl6-nitrobenzothiazole-2-carboxylate is then expected to be reduced to acarboxamide group and then to a cyano group. In the illustration of FIG.6B nitration of C6 of the benzothiazole is performed after the reducingthe carboxylate group in C2 to a carboxamide and before the expectedconversion of the carboxamide group to a cyano group. In theillustration of FIG. 6C nitration of C6 of the benzothiazole isperformed after reducing the carboxylate group in C2 to a carboxamideand before the expected conversion of the carboxamide to a cyano group,followed by a reduction of the C6 position to an amino group.

FIG. 7 shows schematic representation of a method to provide2-cyano-6-aminobenzothiazole from a monofunctional benzothiazoleaccording to an embodiment herein described. In particular, theillustration of FIG. 7 shows synthetic routes consisting of a series ofnitration reactions expected to generate the intermediate derivatives tothe D-aminoluciferin precursor, 2-cyano-6-aminobenzothiazole accordingto embodiments herein described.

FIG. 8 shows a nuclear magnetic resonance spectra for a compound hereindescribed. In particular FIG. 8 shows a nuclear magnetic resonance(¹³C-NMR) spectra of ethyl 6-nitrobenzothiazole-2-carboxylate 7 obtainedon a Bruker 500 MHz machine in d₆-DMSO.

FIG. 9 shows a schematic representation of a method to provide an aminoacid or a peptide labeled with 6-amino-6-deoxy-D-luciferin according toan embodiment herein described. In particular, the illustration of FIG.9, schematically shows a 3-step process of the synthesis of2-cyano-6-aminobenzothiazole, the precursor to6-amino-6-deoxy-D-luciferin, conjugated to single amino acids (R1=one ofthe 20 amino acids) followed by subsequent conjugation to R₂ (R₂=eitheranother amino acid or a peptide sequence) and then cyclization of thering structure to generate bioluminescent probes according toembodiments herein described.

FIG. 10 shows a schematic representation of the two steps depicted inFIG. 9 according to an embodiment herein described. In particular, FIG.10A shows a schematic representation of a conjugation of single aminoacids to 2-cyano-6-aminobenzothiazole, the precursor to6-amino-6-deoxy-D-luciferin (D-aminoluciferin) and subsequent schematicrepresentation of a cyclization of ring structure to form amino acidconjugated 6-amino-D-luciferin. FIG. 10B shows a schematicrepresentation of a method to provide an amino acid or a peptide labeledwith 6-amino-6-deoxy-D-luciferin from a 2-cyano-6-aminobenzothiazoleaccording to an embodiment herein described. In particular, theschematics of FIG. 10B show a generalization of a step-wise process forpreparation of the precursor to D-aminoluciferin comprising firstconjugating a single amino acid (R1), followed by subsequent conjugationwith a longer peptide sequence (R2), and then formation of thepeptide-conjugated D-aminoluciferin substrate via cyclization.

FIG. 11 shows a schematic representation of a method to provide an aminoacid or a peptide labeled with 6-amino-6-deoxy-D-luciferin from amonofunctionalized benzothiazole according to an embodiment hereindescribed

FIG. 12 shows a schematic representation of a method to provide an aminoacid or a peptide labeled with 6-amino-6-deoxy-D-luciferin from a2-cyano-6-aminobenzothiazole according to an embodiment hereindescribed. In particular, the schematics of FIG. 13 show a specificexample of the method schematically illustrated in FIG. 11 where R₁ is atyrosine amino acid and R₂ is a specifically designed biotin-labeledpeptide sequence that can be recognized by the serine protease, prostatespecific antigen. The biotin functional group on the end of the peptidesequence facilitates coupling to a streptavidin compound wherein thisfinal compound could be utilized in an assay system.

FIG. 13 shows a schematic representation of a method to provide an aminoacid or a peptide labeled with 6-amino-6-deoxy-D-luciferin from amonofunctionalized benzothiazole according to an embodiment hereindescribed.

FIG. 14 shows a schematic representation of a method to provide acarboxylate modified luciferin amino acid/peptide probes according to anembodiment herein described.

FIG. 15 shows a schematic representation of a method using a carboxylatemodified luciferin amino acid/peptide probes according to an embodimentherein described.

FIG. 16 shows liquid chromatography/mass spectrometry spectra forcompounds of methods and systems of the disclosure, according to anembodiment herein described.

FIG. 17 shows liquid chromatography/mass spectrometry (FIG. 17A) andnuclear magnetic resonance spectra (FIG. 17B) for a compound accordingto an embodiment herein described.

FIG. 18 shows liquid chromatography/mass spectrometry spectra for acompound according to an embodiment herein described.

FIG. 19 shows liquid chromatography/mass spectrometry (FIG. 19A) andnuclear magnetic resonance spectra (FIG. 19B) for a compound accordingto an embodiment herein described.

FIG. 20 shows liquid chromatography/mass spectrometry spectra for acompound according to an embodiment herein described.

FIG. 21 shows liquid chromatography/mass spectrometry spectra for acompound according to an embodiment herein described.

DETAILED DESCRIPTION

Provided herein are methods and systems to synthesize a precursor of6-amino-6-deoxy-D-luciferin, (herein also D-aminoluciferin), andderivatives thereof, which can be used in connection with variousapplications that are based on the reaction between luciferase enzyme ofthe North American firefly (Photinus pyralis) and substrate D-luciferin.

The reaction between the luciferase enzyme and substrate D-luciferin iswidely used as an optical reporter for in vitro and in vivo assays.Broad ranging applications of the luciferase enzyme as optical reportercomprise reporting gene expression¹, proliferation of cancerous cells,²efficacy of cancer therapies³, bacterial infection⁴ and enzymaticaction.^(5, 6) Under various reaction conditions identifiable by askilled person, photons are generated during the enzymatic conversion ofD-luciferin to oxyluciferin in the presence of compounds such as ATP, O₂and Mg²⁺.⁷⁻⁹ In several cases bioluminescence can be detected with a lowintrinsic background in cells and tissues, sometimes in contrast tofluorescent-based detection, where autofluorescence emits significantbackground noise. Studies to modify the natural substrate D-luciferin innumerous positions have generated substrates with novel properties. Forexample, masking the carboxyl group and/or methylating the 6-positionphenolic group of D-luciferin inhibits reaction with the luciferaseenzyme. Enzymatic or chemical hydrolysis of the carboxylic acid mask anddemethylation of the methoxy group releases the free substrate forsubsequent reaction with the luciferase enzyme.⁶ In various cases6-amino-6-deoxy-D-luciferin, D-aminoluciferin, with an amino group inthe 6-position on the benzothiazole ring,¹⁰ allows conjugation of theoptical reporter substrate to peptide sequences to generate proteaseassay probes.^(5,10,11-13)

FIG. 1 shows a cartoon scheme depicting an exemplary application ofbioluminescent assay performed with labeled probes obtainable with themethods and systems herein described. In particular, in the illustrationof FIG. 1, a complex biological sample (ex. blood sample) wheninteracting with a specifically designed protease probe has thecapability to release the D-aminoluciferin, which ultimately provideslight output in the presence of Mg²⁺ and ATP when proteases (e.g.indicative of disease) are present in the biological sample. In someembodiments, this assay can serve as a marker for disease.

Methods and systems are herein described to provide a D-aminoluciferinprecursor formed by 2-cyano-6-aminobenzothiazole (herein alsoD-aminoluciferin precursor or simply precursor) and/or probes labeledwith D-aminoluciferin provided starting from the precursor.

In some embodiments, methods and systems are described to provide theprecursor with method starting from a monofunctional benzothiazole. Theterm “benzothiazole” as used herein indicates an organosulfur compoundof formula (V)

which can be substituted or unsubstituted in the various positions.Benzothiazole is typically colorless, and takes the form of a slightlyviscous liquid while most of its derivatives are solid. Benzothiazolesare commercially available and can be prepared using methods such astreatment of 2-aminobenzenethiol with acid chlorides according to thereaction C₆H₄(NH₂)SH+RC(O)Cl→C₆H₄(NH)SCR+HCl+H₂O and to additionalprocedures identifiable by a skilled person.

The term “monofunctional benzothiazole” indicates a benzothiazoleincluding one functional group in the C2 position wherein the term“functional group” as used herein indicates a specific group of atomswithin a molecular structure that are responsible for the characteristicchemical interactions or reactions of that structure. In general,exemplary functional groups include hydrocarbons, groups containinghalogen, groups containing oxygen, groups containing nitrogen and groupscontaining sulfur all identifiable by a skilled person. In particular,exemplary functional groups in the sense of the present disclosurecomprise alkoxy or alkyl groups, secondary amines, amides and additionalfunctional groups can be identified by a skilled person upon reading ofthe present disclosure. In some embodiments, the monofunctionalbenzothiazole can be substituted in any positions, wherein the terms“substituted” or “substitution” herein indicates replacement of one ormore hydrogens with chemical groups that do not substantially interferewith the characteristic chemical interactions or reactions of thefunctional group as described herein.

In some embodiments, methods and systems are described to provide theprecursor with methods starting from a monofunctional benzothiazoleattaching in position C2 a functional group of formula (I) (C(═X₁)NH₂)wherein X₁ is O or S, wherein the amide in the functional group offormula (I) is converted to a cyanide group by elimination of a H₂X₁compound.

The term “amide” as used herein indicates an organic compound thatcontains the carbonyl group (R₄—C═O ) linked to a nitrogen atom (N)(herein also carboxamide) or a thiamide group R₄—CS—NR′R₅, where R₄, R′,and R₅ are same or different and are organic groups with R′ and R₅possibly formed by H. The term refers both to a class of compounds and afunctional group within those compounds.

In particular, the term “carboxamide” as used herein indicates anorganic compound that contains a carbonyl group (R4-C═O) linked to anitrogen atom (N). Typically, carboxamides of the general structureR6-CO—NR7R2R8 can be synthesized by replacement of the hydroxyl group(OH) of a carboxylic acid by an amino group (NR1R2), where R6, R7, andR8 are same or different and are organic substituents with R7 and R8possibly formed by H. Exemplary compounds containing carboxamide groupscomprise asparagine and glutamine.

The term thioamides indicates sulfur analogues to amides, where theoxygen atom (O) in amide is replaced by a sulfur atom (S). The generalstructure of thioamide is R₄—CS—NR′R₅, where R₄, R′, and R₅ are same ordifferent and are organic groups with R′ and R₅ possibly formed by H.Typically thioamides are analogous to carboxamides but they exhibitgreater multiple bond character along the C—N bond, resulting in alarger rotational barrier. Thioamides are typically prepared by treatingamides with phosphorus sulfides such as phosphorus pentasulfide and, inmore specialized applications, Lawesson's reagent. An alternative routeentails the reaction of nitriles with hydrogen sulfide. TheWillgerodt-Kindler reaction also affords benzylthioamides.

The terms “eliminate” and “elimination reaction” as used herein withreference to a reaction indicate an elimination reaction. An eliminationreaction is a type of organic reaction in which two substituents areremoved from a molecule in either a one or two-step mechanism (E2 and E1mechanisms, respectively). Either the unsaturation of the moleculeincreases or the valence of an atom in the molecule decreases by two. E1mechanism: generally follows the following form: the first step requiresthe loss of the leaving group, forming a carbocation intermediate. Anucleophilic species, usually a base, then attacks a neighboringhydrogen forming the double bond. E2 mechanism generally follows thefollowing form: a nucleophilic species or base attacks a hydrogenneighboring the leaving group, pushing the electrons into the doublebond as the leaving group leaves. In elimination reactions hereindescribed does not necessarily result in an actual production of an H₂X₁product, as long as the H₂X₁ compound is removed from the startingcompound. In this connection for example, in embodiments where X₁ isoxygen and a POCl₃ reagent is used the oxygen atom is eventuallyattached to phosphorus. Exemplary elimination reactions comprisedehydration and dehydrosulfurization. A dehydration reaction is usuallydefined as a chemical reaction that involves the loss of water from thereacting molecule. Dehydration reactions are a subset of eliminationreactions where the leaving group is water (H₂O). Dehydrosulfurizationreactions or H₂S elimination reactions are another subset of eliminationreaction where the leaving group is (H₂S).

In some embodiments, in the functional group of formula (I) X₁ is S, theconversion from thioamide to CN group can be performed bydehydrosulfurization directed to elimination the H₂S compound. Thedehydrosulfurization can be performed with suitable reagents (e.g.tellurium chloride) and under suitable conditions, such as the onesdescribed for example in ref. 17. A skilled person will be able toidentify additional reactions and related procedures that are functionalto the dehydrosulfurization of the monofunctional benzothiazole hereindescribed.

In some embodiments, in the functional group of formula (I) X₁ is O thereduction can be performed by dehydration of the corresponding estergroup with production of H₂O according to procedures such as the onesexemplified in the Examples section and additional proceduresidentifiable by a skilled person.

In some embodiments, the benzothiazole presenting the functional groupof formula (I) in position C2 and/or the benzothiazole presenting thefunctional group CN comprises an amino group in position C6. In someembodiments, the benzothiazole presenting the functional group offormula (I) in position C2 is modified to comprise an amino group inposition C6. In particular in some of those embodiments, a nitrile groupis first introduced in position C6 before or after converting thefunctional group of formula (I) to a CN group. The nitrile group in C6of the benzothiazole is then converted to an amino group. A skilledperson will be able to identify suitable methods to introduce an NO2group in monofunctional benzothiazoles herein described at any stage ofthe procedures. For example, typical nitrations use nitric acid andsulfuric acid to produces the nitronium ion (NO₂), which is the activespecies of nitration reactions and attacks the electron-rich reactant,such as a benzene ring, to initiate electrophilic substitution reaction.

In some embodiments, an NH2 is introduced directly into the C6 positionof the benzothiazole ring. A skilled reader will be able to identifysuitable reaction for introducing an NH2 group directly inmonofunctional benzothiazoles herein described at any stage of theprocedures herein described.

In some embodiments, the functional group of formula (I) can be providedin the monofunctional benzothiazole herein described starting from amonofunctional benzothiazole attaching in position C2 a functional groupof formula (II) (C(═X₁)X₂R) wherein R is an alkyl group, or a halogenatom and X₁ and X₂ are independently O or S.

The term “alkyl” as used herein refers to a linear, branched, or cyclicsaturated hydrocarbon group typically although not necessarilycontaining 1 to about 10 carbon atoms, preferably 1 to about 6 carbonatoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups suchas cyclopentyl, cyclohexyl and the like. Generally, although again notnecessarily, alkyl groups herein contain 1 to about 6 carbon atoms. Theterm “cycloalkyl” intends a cyclic alkyl group, typically having 4 to 8,preferably 5 to 7, carbon atoms. The term “substituted alkyl” refers toalkyl substituted with one or more substituent groups, and the terms“heteroatom-containing alkyl” and “heteroalkyl” refer to alkyl in whichat least one carbon atom is replaced with a heteroatom. If not otherwiseindicated, the terms “alkyl” and “lower alkyl” include linear, branched,cyclic, unsubstituted, and/or substituted. In some embodiments, in thefunctional group of formula (I), R can be a substituted alkyl which cancomprise heteroatoms such as N, O, S, P, Si, F, Cl, Br, and I, as wellas additional groups and heteroatoms identifiable by a skilled person.

In embodiments where X₂ is O, the resulting X₂—R group can be an alkoxygroup or an acid halide. The term “alkoxy” as used herein intends analkyl group bound through a single, terminal ether linkage; that is, an“alkoxy” group may be represented as —O-alkyl where alkyl is as definedabove. A “lower alkoxy” group intends an alkoxy group containing 1 to 6carbon atoms. Exemplary alkoxy group comprised in the functional groupof formula (I) comprise methoxy, ethoxy, propoxy and additional alkoxygroup comprising a lower alkyl. The term “acid halide” indicates achemical compound that can be typically derived from an oxoacid byreplacing a hydroxyl group with a halide group (i.e. a binary compound,of which one part is a halogen atom and the other part is an element orradical that is less electronegative (or more electropositive) than thehalogen). Exemplary acid halides comprise (C(═O)Cl) and additionalhalides identifiable by a skilled person. In some embodiments, the acylhalide can be treated with ammonia, and the reaction is expected toproceed faster than the reaction performed with the ester group.

In embodiments where X₂ is S the resulting X₂—R group is an alkyl groupand in particular a lower alkyl group, or a halogen such as Cl.Exemplary functional groups of formula (II) comprise (C(═S)OR), whichcan be made for example by treating the ester with P₂S₅ as described inref. 19; (C(═O)SR) which can be made for example by reacting the esterwith a mercaptan under basic conditions as identifiable by a skilledperson; and (C(═S)SR), which can be made for example by treating thechloromethylbenzothiazole with sulfur and methyl iodide, according tothe procedure described in ref. 20. A skilled reader will be able toidentify additional functional groups of formula (II) and suitableprocedure to provide monofunctional benzothiazole comprising thosefunctional groups.

In some embodiments, wherein the functional group of formula (I) and(II) X₁ is S, the conversion from ester to thioamide can be performedusing reagents and procedures identifiable by a skilled person. For theconversion from ester to thioamide can be performed by using NH₃, thenP₂S₅ as described in ref 18. Additional reactions will be identifiableby a skilled person upon reading of the present disclosure.

In some embodiments, wherein the functional group of formula (I) and(II) X₁ is O, the conversion from ester to carboxamide can be performedby using amines alone or in presence of reagents (such asdicyclohexylcarbodiimide (DCC)) which are suitable to convert thehydroxyl oxygen in a more efficient leaving group. Examples of thoseconversions are reported in the Example section of the presentdisclosure. A skilled reader will be able to identify additionalreactions using amines or other reagents that are suitable to convert anester according to formula (I) wherein X₁ is oxygen into a carboxamideof formula (II) wherein X₁ is oxygen.

In some embodiments of methods and systems wherein the D-aminoluciferinprecursor is provided from a monofunctional benzothiazole, themonofunctional benzothiazole is 6-nitrobenzothiazole and the precursorcan be synthesized according to a method schematically illustrated inFIG. 2. In particular, in the illustration of FIG. 2 production of theprecursor can be performed in four steps. In particular a thio orcarboxy ester functional group of formula (II) can be introduced in C2with suitable reactions. The thio or carboxy ester can then be convertedinto a corresponding amide which can then be converted into a nitrile.The NO2 group in C6 can then be reduced to NH2 using procedureidentifiable by a skilled person. Possible variations of this reactionscheme comprise a three steps procedure performed starting from amonofunctional benzothiazole attaching an ester of formula (II), threesteps procedure performed starting from a monofunctional benzothiazoleattaching an amide of formula (I) and other variations wherein thereduction of the NO2 group in C6 is performed before the final reductionof the amide of formula (I) to a CN group.

In an embodiment, wherein X₁ is O the method can be performed asschematically illustrated in FIG. 3. In particular a free radical methodcan be used to generate 2-carboxyethylester-6-nitrobenzothiazole fromcommercially available 6-nitrobenzothiazole according to proceduresexemplified in Example 1.

In the exemplary illustration of FIG. 3, the process further comprisesconverting the 2-carboxyethylester-6-nitrobenzothiazole into acarboxamide which in some embodiments can be carried out using suitablereagents to introduce nitrogen in the functional group of formula (II)which are identifiable by a skilled person upon reading of the presentdisclosure. Exemplary procedures using reagents such as potassium amideor tritylamine and use a diverse range of catalysts such as variousforms of palladium. In an exemplary procedure, nitrogen is introduced byadding ammonia gas in methanol with 100% conversion (TLC and analyticalHPLC) (see Example 2.

In the exemplary illustration of FIG. 3, the6-nitrobenzothiazole-2-carboxamide is then converted into a2-cyano-6-nitrobenzothiazole. In some embodiments, this conversion canbe performed by dehydration of the carboxamide to a nitrile which can becarried out for example using POCl₃ in pyridine, again in quantitativeyield (TLC and analytical HPLC). The corresponding nitrile can then bepurified using gravity column silica chromatography before the finalstep and starting material 6-nitrobenzothiazole can be recovered (seeExample 3). Additional procedures to perform dehydration compriseprocedures which use silanes, zirconium catalysts, palladium catalysts,phosphines according to techniques identifiable by a skilled person.Additional procedures and techniques which are also suitable for theconversion of carboxamide to a nitrile are also identifiable by askilled person.

In the exemplary illustration of FIG. 3, the reduction of the NO2 groupin C6 to a NH2 is performed following conversion of the C2 into anitrile group as a final reaction to generate the6-amino-6-deoxy-D-luciferin, D-aminoluciferin, precursor. The reductionof NO2 in C6 can be performed using methods identifiable by a skilledperson, such as, procedures performed using stannous chloride in ethanolexemplified in the Example section. In those reactions, a sufficientnumber of equivalents of the reductant (e.g. about 2.5 to about 5 eq)can be used to reduce the nitro group in a predetermined amount of time(e.g. about 2 h). Silica chromatography purification can also be used toyield D-aminoluciferin precursor with high purity for subsequentcoupling reactions (e.g. to amino acids and peptides) (see Example 4).

In some embodiments according to the illustration of FIG. 3, reactionsare selected so that subsequent conversions of ethyl6-nitrobenzothiazole-2-carboxylate are not impeded by a mixture of ethyl6-nitrobenzothiazole-2-carboxylate and 6-nitrobenzothiazole, andtherefore, the mixture can be carried forward without purification. Insome of those embodiments, the synthesis is particularly suitable foractual production of material for use in assays, kits, and additionalapplications identifiable by a skilled person since purification is notnecessary.

A skilled person will be able to identify variants of the procedureschematically illustrated in FIG. 3 with other functional groups offormula (I) and (II) and with reduction of the NO₂ in C6 position atdifferent stages before the conversion of the functional group in C2upon reading of the present disclosure as also exemplified by theExamples section and figures.

For example additional exemplary reaction schemes starting from adifferent monofunctional benzothiazole herein described, areschematically illustrated in FIG. 5 which shows a specific functionalgroup of formula (II) wherein X₁ and X₂ are oxygen and R is an ethylgroup. In the exemplary illustration of FIG. 5, the startingmonofunctional benzothiazole is ethyl benzothiazole-2-carboxylate andthe nitration of C6, and conversion of the ethyl ester C2 group into acarboxamide and then to a nitrile group can be performed according toany of the procedure herein described.

Some specific routes are further illustrated in FIGS. 6 and 7. Inparticular, FIGS. 6 and 7, show specific schemes for alternativesynthesis of 2-cyano-6-aminobenzothiazole starting with a commerciallyavailable 2-carboxyethyl ester. As shown in the exemplary illustrationof FIG. 6, it is expected that nitration can be performed at any pointand the possibility to nitrate either the ester (FIG. 6A) or thecarboxamide (FIG. 6B) has been demonstrated.

In the exemplary illustration of FIG. 6A, the monofunctionalbenzothiazole is ethyl benzothiazole-2-carboxylate and the2-cyano-6-aminobenzothiazole can be provided following nitration andsubsequent reduction of the benzothiazole. Nitration of the commerciallyavailable, ethyl benzothiazole-2-carboxylate was also carried out usingconcentrated H₂SO₄ and KNO₃ resulting in a mixture of2-carboxyethylester-6-nitrobenzothiazole with starting material (TLC).Gravity silica column chromatography of2-carboxyethylester-6-nitrobenzothiazole proved challenging to separatefrom starting material 2-carboxytheylester-benzothiazole (10% yield).(see Example 5).

In the exemplary illustration of FIGS. 6B and 6C, converting the ethylbenzothiazole-2-carboxylate into a 2-carboxamide-benzothiazole iscarried out with ammonia (e.g. using ammonia gas in methanol with 100%conversion (TLC, analytical HPLC) see Example 6). Without purification2-carboxamide-benzothiazole can be reacted (e.g. with conc. H₂SO₄ andHNO₃) to yield a mixture of 6-nitrobenzothiazole-2-carboxamide and2-carboxamide-nitrobenzothiazole (˜50% by TLC) (see Example 7). Suitabletechniques can be used to separate the mixture (for example gravitysilica column chromatography of 6-nitrobenzothiazole-2-carboxamide (˜38%yield) see Example 7).

In some embodiments, converting the 2-carboxamide-benzothiazole to acorresponding nitrile in C2 position can be performed eliminationreactions and additional reactions identifiable by a skilled person. Inthe methods herein described for example conversion of the C2carboxamide can be performed by dehydration to the nitrile usingsuitable reagents such as phosphoryl chloride in pyridine, proceedingwith quantitative conversion. In some of those embodiments,benzothiazole-2-carboxamide can be converted to the nitrile to yield2-cyano-benzothiazole with 100% conversion (analytical HPLC, mass spect)(see Example 8). Without purification, the nitrile was reacted withH₂SO₄/HNO₃ and the reaction was monitored over time (over a period of ˜2h) via analytical HPLC. (see Example 11).

A skilled person will be able to identify alternative reactions schemesaccording to the illustration of FIGS. 5 to 7, wherein in the functionalgroup (II) of the monofunctional benzothiazoles, X₁ and/or X₂ are Sand/or R is provided by a different alkyl group (in particular a loweralkyl group) or a halogen (e.g. Cl), upon reading of the presentdisclosure. In any of those embodiments wherein the monofunctionalbenzothiazole used in methods and systems herein described does notinitially comprise a nitro or amino group in position C6, the aminogroup is introduced at various stages as will be understood by a skilledperson. For example in some embodiment, it is expected that the NO₂group can be introduced using concentrated nitric and sulfuric acids,giving regio selectivity for the C6 position over the C4,C5, or C7positions of the benzothiazole ring presenting a carboxamide, beforeconverting the carboxamide to a cyanide group. In some embodiments, theNH2 group can be introduced at any stage with other reactions which donot require nitration of the C6 position that will be identifiable by askilled person upon reading of the present disclosure.

In an embodiment of methods and systems provide the D-aminoluciferinprecursor the monofunctional benzothiazole is 6-nitrobenzothiazole. Insome of those embodiments cyanation of the 2-position of6-nitrobenzothiazole can be performed by forming a lithium salt ofbenzothiazole with butyllithium then reacting this with a solublecyanide followed by quenching of excess butyl lithium with water¹⁵.Starting material and only a small amount of6-nitrobenzothiazole-2-carbonitrile resulted. Direct cyanation of the C2position of other monofunctional benzothiazoles herein described can beperformed according to techniques and procedures identifiable by askilled person upon reading of the present disclosure.

In some embodiments, the 6-amino-6-deoxy-D-luciferin, D-aminoluciferin,precursor obtained with any of the methods and/or systems hereindescribed or provided using other methods can be coupled to an aminoacid or peptide labeled to provide an amino acid-conjugated or a peptideconjugated-2-cyano-6-aminobenzothiazole.

As used herein the term “amino acid”, “amino acid monomer”, or “aminoacid residue” refers to any of the twenty naturally occurring aminoacids including synthetic amino acids with unnatural side chains andincluding both D and L optical isomers. The term “polypeptide” or“peptide” as used herein indicates an organic polymer composed of two ormore amino acid monomers and/or analogs thereof. The term “polypeptide”includes amino acid polymers of any length including full lengthproteins and peptides, as well as analogs and fragments thereof. Apolypeptide of three or more amino acids is also called a proteinoligomer or oligopeptide. The term “amino acid analog” refers to anamino acid in which one or more individual atoms have been replaced,either with a different atom, isotope, or with a different functionalgroup but is otherwise identical to its natural amino acid analog.

The term “couple” or “conjugate” as used herein indicates formation of acovalent bond between two compounds. Typically, in some embodiments,coupling or conjugation of the amino acid or to the amino group in2-cyano-6-aminobenzothiazole can be performed with an efficient peptidecoupling method such as DCC, or N-methyl morpholine andisobutylchloroformate, since the aniline amino group is relativelyunreactive. In other embodiments, coupling or conjugation can beperformed reactions suitable to form a urea/thiourea (NH(C═X)NH) or acarbamate/thiocarbamate (X(C═X)NH) linkage, where X═O or S.

The term “carbamate” as used herein indicates organic compounds derivedfrom carbamic acid (NH2COOH). Specifically, the term refers to any saltor ester of carbamic acid with the general structure R8O—CO—NR9R10,where R8, R9, and R10 are same or different and are organic substituentswith one or two possibly formed by H. The term “thiocarbamate” as usedherein indicates sulfur analogues of carbamates, where one of the oxygenatoms (O) in a carbamate is replaced by a sulfur atom (S). Specifically,there are two structurally isomeric types of thiocarbamates:O-thiocarbamates, ROC(═S)NR2, where the carbonyl group (C═O) is replacedwith a thiocarbonyl group (C═S) and S-thiocarbamates, RSC(═O)NR2, wherethe R—O—group is replaced with an R—S— group. Further, O- andS-thiocarbamates can interconvert, for example in the Newman-Kwartrearrangement.

In particular, in some embodiments coupling of the peptide with theprecursor can be performed according to a two step process schematicallyillustrated in FIGS. 9 and 10 wherein a process is illustrated thatcomprises conjugating an amino acid and/or a peptide to the precursor.In particular, in some embodiments the conjugation between amino acidand/or peptide can be performed in a two step reaction wherein the aminoacid or peptide is first conjugated with the peptide and then theresulting amino acid-conjugated or peptide-conjugated precursor isreacted with cysteine to provide an amino acid-conjugatedD-aminoluciferin or a peptide-conjugated D-aminoluciferin (FIG. 10A). Inother embodiments, a three step reaction is performed wherein the anamino acid-conjugation with the precursor is performed, followed byconjugation with the specific peptide sequence, and then reacting withD-cysteine to provide the peptide-conjugated D-aminoluciferin (FIG. 10B)

In some embodiments, conjugation of the amino group of the2-cyano-6-aminobenzothiazole to single amino acids or peptides can beachieved using isobutylchloroformate activation of the free acid of theamino acid in the presence of N-methyl morphiline in THF as previouslyreported.^(11, 13) (see Example 10). In some embodiments, conjugation ofthe amino group of the 2-cyano-6-aminobenzothiazole to single aminoacids can be achieved using carbodiimide activation. In particularcarbodiimide activation involves attack of the carboxylate oxygen on thecentral carbodiimide carbon, creating a leaving group with that oxygenatom according to reactions identifiable by a skilled person. The aminethen attacks the carboxyl carbonyl, displacing the now urea leavinggroup. The insolubility of some ureas can in some cases help drive thereaction.

In some embodiments, suitable amino acids to be conjugated with theD-aminoluciferin precursor comprise the 20 naturally occurring aminoacid amino acids, including Isoleucine, Alanine, Leucine, Asparagine,Lysine, Aspartic Acid, Methionine, Cysteine, Phenylalanine, GlutamicAcid , Threonine, Glutamine, Tryptophan Glycine, Valine, Proline,Serine, Tyrosine, Arginine, Histidine, and additional amino acids suchas Selenocysteine Ornithine, and Taurine. In particular, in someembodiments, the amino acids comprise in particular Alanine, Valine,Isoleucine, Leucine, Tyrosine, Methionine, Threonine. In someembodiments, suitable amino acids to be conjugated with theD-aminoluciferin precursor comprise in particular Phenylalanine,Histidine and Glutamic Acid. In some embodiments, amino acids comprisinga side chain (hydrophobic, hydrophilic, acid or basic in nature) can becoupled with the precursor and then cyclized with D-cysteine to yieldthe amino acid-conjugated 6-amino-6-deoxy-D-luciferin, which it isexpected to contribute to varying pharmacokinetics/pharmacodynamicsbehaviors as measured by bioluminescence.

In some embodiments, the amino acids comprise a side chain, which havebeen surprisingly found to be labelable with the precursor and thenconverted in to functional amino acid labeled with D-luciferin despitethe expected modifications in biodistribution(pharmacokinetics/pharmacodynamics) as measured by bioluminescence Inparticular in some embodiments, the amino acid is an amino acid having ahydrophobic side chain (including for example Alanine, Isoleucine,Leucine, Methionine, Phenylalanine, Tryptophan, Tyrosine and Valine). Insome embodiments, the amino acid is an amino acid having a non-polaruncharged side chain (including for example Serine, Threonine,Asparagine and Glutamine). In some embodiments, the amino acid is anamino acid having an electrically charged side chain (including forexample Arginine, histidine and Lysine, Aspartic acid and Glutamicacid).

In some embodiments, an intermediate in the synthesis of peptide labeledat the carboxy terminus with 6-amino-6-deoxy-D-luciferin formed by asubstituted 2-cyano-6-aminobenzothiazole can be provided.

In some embodiments, methods and systems to provide a peptide labeled atthe carboxy terminus with 6-amino-6-deoxy-D-luciferin from amonofunctional benzothiazole. In some embodiments, the peptide can be ofat least 6 or 7 amino acid resides in length and in some of thoseembodiments the system can comprise a further label such a biotin labelor a linker possibly attached and in particular conjugated to thepeptide at the terminus opposite to the terminus where the D-aminoluciferin is conjugated directly or indirectly through conjugation to anamino acid-conjugated D-aminoluciferin precursor. Without any intentionof being limiting and for guidance purpose only it is expected that useof the three step process schematically illustrated in FIG. 10B will beparticularly useful for synthesis of a peptide with a sequence of 5amino acid residues or longer and/or to a peptide further labeled withbiotin or other labels. In particular, in some of those embodiments, theD-aminoluciferin precursor is then first reacted with an amino acid andthen redirectly with a long peptide chain thus allowing conjugation whenthe amino group on the precursor is not reactive enough to directlyconjugate the peptide chain in an efficient manner according to anexperimental design.

In an embodiment, a method and system to provide a peptide labeled with6-amino-6-deoxy-D-luciferin is described. The method comprisesconjugating the peptide with an amino acid-conjugated2-cyano-6-aminobenzothiazole to provide a peptide-conjugated2-cyano-6-aminobenzothiazole, the conjugating performed to allowformation of a peptide bond between the amino group of the aminoacid-conjugated 2-cyano-6-aminobenzothiazole and a carboxylic group ofthe peptide. The method further comprises reacting thepeptide-conjugated 2-cyano-6-aminobenzothiazole with D-cysteine toprovide a peptide-conjugated 6-amino-6-deoxy-D-luciferin. The systemcomprises one or more peptides, peptide-conjugated6-amino-2-cyanobenzothiazole and/or amino acid-conjugated2-cyano-6-aminobenzothiazole for simultaneous combined or sequential usein the method to provide a peptide labeled with6-amino-6-deoxy-D-luciferin herein described.

In some embodiments, modification of D-luciferin, 6-hydroxy-D-luciferin,to D-aminoluciferin, 6-amino-6-deoxy-D-luciferin, is expected to resultin substrates that generate novel bioluminescent properties and offersthe opportunity to expand the range of bioluminescence-based assays.D-aminoluciferin, with an amino group in the C6-position of thebenzothiazole ring, allows conjugation of the optical reporter substrateto single amino acids and/or peptide sequences to generate novel assayprobes with the peptide sequences targeting proteases.

In some embodiments, methods and systems herein described provide analternate route for the synthesis of the precursor2-cyano-6-aminobenzothiazole to D-aminoluciferin has been identified andused to generate material for subsequent conjugation to amino acids andpeptide sequences for thorough investigation of the utility ofbioluminescent protease probes.

In some embodiments, methods and systems herein described provideconvenient synthetic route to D-aminoluciferin derivatives of peptides.In particular in some embodiments aminoluciferin derivatives hereindescribed can be used in conjunction with firefly luciferase to reporton cellular events in vivo as well as in bioluminescent assays. Inparticular, in some embodiments, the synthetic route begins with amonofunctional benzothiazole (either 6-nitrobenzothiazole or ethylbenzothiazole-2-carboxylate, proceeds through2-cyano-6-aminobenzothiazole, and results in an amino acid or peptideconjugated to 6-amino-6-deoxy-D-luciferin.

In some embodiments a carboxylate-modified Luciferin, amino acid orpeptide probe, is described, that comprises a D-luciferin moleculeconjugated with the amino-terminus of an amino acid or peptide at thecarboxyl group of said D-luciferin. In some of those embodiments, thecarboxyl-terminus of the amino acid residue or peptide is blocked with ablocking group, such as an amide.

The carboxylate-modified luciferin, amino acid or peptide probe can beprovided using methods and systems based on reactions performed on aresin base. In particular the amino acid or peptide can be provided on aresin base (e.g. by synthesizing the peptide on the resin base). AD-luciferin molecule can then be conjugated at the amino terminus of thepeptide or amino acid on the resin bases via formation of a peptidebond. The resulting conjugate can be cleaved from the resin to produce aproduce free carboxylate-modified luciferin, amino acid/peptide probethat can then be optionally purified if desired. In some embodiments theresin base is a chlorotrityl chloride (CTC) resin. In some embodimentssynthesizing the peptide can be performed using standard Fmoc amino acidsynthesis. Purification can be performed with techniques identifiable bya skilled person, for examples purification can be using reverse phasehigh performance liquid chromatography. Following purification thecarboxyl-terminus of the said synthesized probe can be blocked using ablocking agent such as an amide.

The carboxylate-modified Luciferin, amino acid or peptide probe can beused to perform various assays and procedure aimed at detection oftargets and/or reactions. For example in some embodiments thecarboxylate modified luciferin amino acid or peptide can be used inconnection with luciferase for releasing the D-luciferin molecule in theassay.

In particular, in some embodiments, carboxylate-modified luciferinprobes are provided, where D-luciferin is modified at the carboxylicacid with amino acids/peptides to inhibit the reaction with theluciferase enzyme. In some of those embodiments, upon carboxypeptidasedigestion of the amino acids conjugated to the D-luciferin, the freebioluminescent substrate is released for subsequent reaction with theluciferase enzyme. Further, in some embodiment, if the carboxyl-terminusof a D-luciferin-peptide sequence is blocked as a carboxamide or othersuitable blocking agent, only upon proteolytic action is a freecarboxylic acid generated for carboxypeptidase to begin digestion torelease the D-luciferin. Combined together, these concepts are used togenerate carboxylate-modified luciferin, amino acid/peptide probes andassays using such probes or any derivative thereof to monitor the actionof proteases such as endoproteases or exopeptidase of interest.

The term “derivative” as used herein with reference to a first compound(e.g., D-aminoluciferin), indicates a second compound that isstructurally related to the first compound and is derivable from thefirst compound by a modification that introduces a feature that is notpresent in the first compound while retaining functional properties ofthe first compound. Accordingly, a derivative of D-aminoluciferin,usually differs from the original D-aminoluciferin by modification ofthe substituents of the D-aminoluciferin that might or might not beassociated with an additional function not present in the originalD-aminoluciferin. The additional function, and the properties associatedwith D-aminoluciferin can be verified with methods and systems such asthe ones described in the Examples section in connection with exemplaryprocesses and derivatives herein described.

In some embodiments, derivatives of D-aminoluciferin herein describedcan be used in connection with several assays directed to in vitro or invivo detection of targets and/or reactions.

The term “detect” or “detection” as used herein indicates thedetermination of the existence, presence or fact of a target or signalin a limited portion of space, including but not limited to a sample, areaction mixture, a molecular complex and a substrate including aplatform and an array. In particular, D-aminoluciferin typicallyprovides bioluminescence which is an optically detectable signal.

In some embodiments, the D-aminoluciferin derivatives herein describedcan be used in assays where bioluminescent detection has extremely highsensitivity because the low background interference is not affected bycellular auto-fluorescence and other fluorescent contaminants and doesnot require extreme operation conditions. An example is provided byprotease assays based on bioluminescence in connection with in vitro andin vivo applications which are identifiable by a skilled person.Proteases can be specific or non-specific in their action and therefore,a wide range of protease substrates may be required to characterizeprotease proteomics in different complex backgrounds (e.g. in vivo orserum). A wide variety of proteases are present in all living organismsand in protease activity levels are often a part of the host response toinfection or disease.¹⁶ Therefore, monitoring protease activity in bothin vitro and in vivo experiments is potentially an early warning systemfor a particular diseased state before clinical symptoms occur.

Exemplary suitable assays are provided by in vitro assays for certainprotease developed with different reporting techniques. The most commontechniques are the proteolytic release of p-nitro aniline for UVdetection, the proteolytic release of fluorescence dyes for fluorescencedetection and mass spectrometry detection of proteolytic cleavageproducts. However, there are various problems associated with thedetection techniques currently available when developing an assay fordetecting specific reactions in a complex background (e.g.in vivo—thelow sensitivity of UV absorption, auto-fluorescence of cells orfluorescent interference from chemical and natural products, and thevacuum and other expensive and cumbersome parts required for massdetection).

In methods and systems herein described, any of the above compounds canbe synthesized or added according to techniques identifiable by askilled person.

As disclosed herein, the compounds herein described can be provided as apart of systems to detect targets according to any of the methodsdescribed herein. The systems can be provided in the form of kits ofparts.

In a kit of parts, the monofunctionalized benzothiazoles, reducingagents, and other reagents to perform the methods can be comprised inthe kit independently. One or more compounds and reagents can beincluded in one or more compositions alone or in mixtures identifiableby a skilled person. Each of the one or more of compounds and reagentscan be in a composition together with a suitable vehicle.

Additional reagents can include salts (such as Mg²⁺) and reagents (e.g.ATP) and/or molecules suitable to enhance or favor the reactionaccording to any embodiments herein described and/or molecules,standards and/or equipment to allow detection of pressure temperatureand possibly other suitable conditions. For example luciferin/luciferaseas control reagents can be comprised in a kit to serve as the “parentcompounds” that will provide the baseline for comparison to the modifiedbioluminescence substrates.

In particular, the components of the kit can be provided, with suitableinstructions and other necessary reagents, in order to perform themethods here described. The kit will normally contain the compositionsin separate containers. Instructions, for example written or audioinstructions, on paper or electronic support such as tapes or CD-ROMs,for carrying out the assay, will usually be included in the kit. The kitcan also contain, depending on the particular method used, otherpackaged reagents and materials (i.e. wash buffers and the like).

Further advantages and characteristics of the present disclosure willbecome more apparent hereinafter from the following detailed disclosureby way of illustration only with reference to an experimental section.

EXAMPLES

The methods system herein described and related intermediates andderivatives are further illustrated in the following examples, which areprovided by way of illustration and are not intended to be limiting.

In particular, the following examples illustrate exemplary synthesis anduses of 2-cyano-6-aminobenzothiazole starting from 6-nitrobenzothiazole,ethyl 6-nitrobenzothiazole-2-carboxylate, and other compounds performedaccording to the reaction schemes summarized in FIGS. 1-13. A personskilled in the art will appreciate the applicability of the featuresdescribed in detail for 2-cyano-6-aminobenzothiazole for additionalcompounds (e.g. additional monofunctional benzothiazole) having same ordifferent chemical characteristics according to the present disclosure,and to related derivatives. The following examples also illustratedexemplary synthesis and uses of a carboxylate modified luciferin aminoacid probe for bioluminescent protease assay according to reactionsschemes summarized in FIGS. 14 and 15. A skilled person will appreciatethe applicability of the features described in detail for the specificphenylalanine probe shown in the examples to other probes according tothe present disclosure.

The following experimental procedures and characterization data wereused for all compounds and their precursors exemplified herein.

General. Commercially available reagents and solvents were used asreceived without further purification. Anhydrous pyridine and phosphorylchloride were purchased from Aldrich. 6-nitrobenzothiazole was purchasedfrom Alfa Aesar. Anhydrous ammonia was purchased from Mattheson Tri-Gas,ethyl pyruvate from Fluka, ferrous sulfate from Mallinckrodt Chemicals,sodium bicarbonate from EMD Biosciences, and tin(II) chloride fromSigma. The amino acid compounds were purchased from NovaBioChem and thebiotin-conjugated peptides were specifically designed and purchased fromPeptides International.

Characterization/Instrumentation. Analytical thin layer chromatography(TLC) was carried out using aluminum sheets coated with silica gel 60F₂₅₄. Reaction conversions were followed by analytical HPLC at 1 mL/minon an Agilent 1100 machine (Waters Symmetry C18, 5 μm, 4.2×150 mmcolumn, diode array detector) with a linear gradient from 95% H₂O (0.1%TFA) to 80% MeCN (0.1% TFA) over 15 mi. For analytical characterization,small portions were purified by semi-preparative HPLC at 10 mL/min on aWaters preparative machine (Waters Symmetry prep C18, 7 μm, 19×300 mmcolumn, photodiode array detection) with a linear gradient from 90-95%H₂O (0.1% TFA) to 50-90% MeCN (0.1% TFA) for 30-35 min. Semi-preparativeHPLC fractions were collected and lyophilized using Kinetics Flexi-Dryfreeze-dryer. Nuclear magnetic resonance (NMR) spectra were obtained ona Bruker 500 MHz machine in d₆-DMSO. Splitting patterns are denoted s,singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet;br s, broad singlet. Mass spectra were acquired on a Micromass QuattroMicro API mass spectrometer operating in positive ion mode. The sampleswere dissolved in MeCN/H₂O (1:1), 0.1% formic acid for mass spectrometryanalysis.

EXAMPLE 1 Synthesis of ethyl 6-nitrobenzothiazole-2-carboxylate (2)

Ethyl 6-nitrobenzothiazole-2-carboxylate was synthesized from6-nitrobenzothiazole according to the following reaction scheme.

In particular, 6-nitrobenzothiazole 1 (16.7 mmol, 3.0 g) was suspendedin 6.6 ml deionized H₂O. 2.7 ml conc. H₂SO₄ was added dropwise to thereaction flask. Separately, 30% H₂O₂ (167.6 mmol, 5.7 g) was addeddropwise to a solution of ethyl pyruvate (75.6 mmol, 8.4 ml) at 0° C.The resultant oxyhydroperoxide solution and a solution of FeSO₄.7H₂O(48.9 mmol, 13.6 g) in 13.2 ml deionized H₂O were simultaneously addeddropwise to the 6-nitrobenzothiazole reaction flask at 0° C. After 30min, the reaction was poured onto ice and basified with NaHCO₃ (pH from2 to ˜pH 6). The organic product 2 was washed with a saturated NaClsolution, extracted with EtOAc, dried over Na₂SO₄, and concentrated invacuo. The material was additionally filtered with 200 proof EtOHyielding a pale yellow solid (˜61% yield crude material). Agilent HPLCdisplayed partial conversion to the crude product at 10.4 min with apeak at 8.7 min corresponding to the starting material.

Compound 2 was used in the subsequent step without purification. Foranalytical characterization, a small portion was purified bysemi-preparative HPLC at 10 mL/min on a Waters preparative machine(Waters Symmetry prep C18, 7 μm, 19×300 mm column, photodiode arraydetection) with a linear gradient from 95% H₂O (0.1% TFA) to 90% MeCN(0.1% TFA) for 35 min yielding a large product peak at 27 min. Thefractions were collected and lyophilized using Kinetics Flexi-Dryfreeze-dryer. ESI-MS: m/z calcd for C₁₀H₈N₂O₄S (M+H)⁺ 253.26, found252.85 (100%). ¹H (d₆-DMSO) δ (ppm)=1.39 (t, J=7.0 Hz, 3H), 4.48 (q,J=7.0 Hz, 2H), 8.44 (m, 2H), 9.32 (s, 1H). ¹³C NMR (d₆-DMSO) δ(ppm)=13.8, 63.0, 120.1, 122.1, 125.5, 136.5, 145.9, 155.9, 159.3,164.1.

Bernadi et al reported almost quantatative conversion of benzothiazoleto ethyl 6-nitrobenzothiazole-2-carboxylate.¹⁴ In this case, the lowsolubility of starting material 1 in aqueous H₂SO₄ limited theconversion to the desired product (˜40% by TLC). The product 2 was alsoimpossible to separate from the starting material 1 either by flash orgravity silica column chromatography.

EXAMPLE 2 Synthesis of 6-nitrobenzothiazole-2-carboxamide (3)

6-Nitrobenzothiazole-2-carboxamide was synthesized from ethyl6-nitrobenzothiazole-2-carboxylate according to the following reactionscheme.

In particular, crude ethyl 6-nitrobenzothiazole-2-carboxylate 2 (7.9mmol, 2.0 g) was dissolved in 140 ml MeOH and purged with NH₃ gas.Conversion to the product was monitored by analytical TLC (3 Hexane: 2EtOAc). Following complete conversion to the product (20 min) thesolvent was removed in vacuo. The crude material was filtered withchloroform (30 ml) to yield a pale tan-colored solid (80% yield crudematerial). Agilent HPLC displayed partial conversion to the crudeproduct 3 at 8.1 min. The compound 3 was used in the subsequent stepwithout purification.

For analytical characterization, a small portion was purified bysemi-preparative HPLC at 10 mL/min on a Waters preparative machine(Waters Symmetry prep C18, 7 μm, 19×300 mm column, photodiode arraydetection) with a linear gradient from 90% H₂O (0.1% TFA) to 50% MeCN(0.1% TFA) for 30 min yielding a large product peak at 22.5 minutes. Thefractions were collected and lyophilized using Kinetics Flexi-Dryfreeze-dryer. ESI-MS: m/z calcd for C₈H₅N₃O₃S (M+H)⁺ 224.22, found223.88 (100%). ¹H (d₆-DMSO) δ (ppm)=8.30 (br s, NH), 8.31 (dd, J=9.0 Hz,J=0.5 Hz), 8.41 (dd, J=9.0 Hz, J=2.5 Hz), 8.65 (br s, NH), 9.28 (d,J=2.0 Hz). ¹³C NMR (d₆-DMSO) δ (ppm)=120.3, 122.1, 124.8, 137.1, 145.6,156.6, 160.8, 171.1.

EXAMPLE 3 Synthesis of 2-cyano-6-nitrobenzothiazole (4)

2-Cyano-6-nitrobenzothiazole was synthesized from6-nitrobenzothiazole-2-carboxamide according to the following reactionscheme.

In particular, crude 6-nitrobenzothiazole-2-carboxamide 3 (4.5 mmol, 1.0g), was dissolved in anhydrous pyridine (0.74 mol, 60 ml) and stirred atRT under N₂. The temperature of the reaction was dropped to 0° C. andPOCl₃ (0.14 mol, 12.5 ml) was added dropwise to the reaction flask.After 20 min, the acetone/ice bath was removed and the reaction wasstirred at RT for an additional 2 h. The contents were then transferredto a larger reaction flask containing 150 ml EtOAc at 0° C. Whilestifling, the reaction was quenched with the dropwise addition of water(150 ml).

The organic layer was separated, dried over Na₂SO₄, and concentrated invacuo yielding an orange/brown solid material. TLC analysis (3 Hexane: 7CH₂Cl₂) displayed 100% conversion to the product 4. Agilent HPLCdisplayed conversion to the crude product at 10.1 min. The product 4 waspurified by gravity silica column chromatography (3 Hexane: 7 CH₂Cl₂) toyield a pale white solid (˜24% pure product). ESI-MS: m/z calcd forC₈H₃N₃O₂S (M+H)⁺ 206.20, found 205.97 (100%). ¹H (d₆-DMSO): δ (ppm)=8.47(s, 2H, 1H), 9.39 (s, 1H). ¹³C NMR (d₆-DMSO): δ (ppm)=112.7, 120.3,122.7, 125.3, 136.0, 143.2, 146.4, 154.6.

EXAMPLE 4 Synthesis of 2-cyano-6-aminobenzothiazole (5)

2-Cyano-6-aminobenzothiazole was synthesized from2-cyano-6-nitrobenzothiazole according to the following reaction scheme.

In particular, purified 2-cyano-6-nitrobenzothiazole 4 (0.49 mmol, 100mg) was dissolved in 5 ml EtOH (200 Proof). 2.5 equivalents of SnCl₂(1.3 mmol, 322 mg) was added to the flask. The reaction was heated to60° C. under N₂, and stirred for 2 h. Over time the mixture changed froma bright fluorescent yellow to an orange/yellow coloring. After coolingto room temperature (RT), the reaction was poured into ice water (˜5 ml)and the pH was adjusted using NaHCO₃ (pH 7).

The product 5 was extracted using EtOAc, dried over MgSO₄ and removed invacuo (˜65% yield). Agilent HPLC displayed conversion to the crudeproduct at 7.4 min. The product 5 was purified by gravity silica columnchromatography (3 Hexane: 2 EtOAc) to yield an orange/yellow solid.ESI-MS: m/z calcd for C₈H₅N₃S (M +H)⁺ 176.22, found 175.85 (100%). ¹HNMR (d₆-DMSO, 500 MHz) δ (ppm)=6.15 and 6.55 (br s, NH₂), 6.97 (dd,J=9.0 Hz, J=2.0 Hz), 7.15 (d, J=2.5 Hz), 7.86 (d, J=9.0 Hz). ¹³C NMR(d₆-DMSO) δ (ppm)=102.2, 114.1, 117.3, 125.0, 127.7, 138.2, 143.2,150.4.

EXAMPLE 5 Synthesis of ethyl 6-nitrobenzothiazole-2-carboxylate (7)

Ethyl 6-nitrobenzothiazole-2-carboxylate was synthesized from ethylbenzothiazole-2-carboxylate according to the following reaction scheme.

In particular, commercially available ethyl benzothiazole-2-carboxylate6 (24.1 mmol, 5.0 g) was suspended in 22 ml conc. H₂SO₄ at 0° C. At 10°C. KNO₃ (26.4 mmol, 26 g) was added portionwise over 30 min to thestifling solution, not to exceed 15° C. Color of reaction changed frombright green/yellow to yellow over time. The reaction warmed from 25° C.to 40° C. for an additional 30 min. When the temperature of the reactionwas decreased, the solution was poured over ice/water). The precipitateforming in solution was collected via glass frit vacuum filtration toyield a light yellow solid 7.

Resultant solid 7 was additionally washed with water, extracted intoEtOAc, and concentrated in vacuo. Agilent HPLC displayed partialconversion to the crude product at ˜10.0 min with a peak at ˜9.9 mincorresponding to the starting material. Attempts to purify the productwere made using gravity silica column chromatography (4 Hexane: 1 EtOAc)to yield a yellow solid (˜10% yield). Compound 7 was identified withLC/MS spectroscopic analysis as illustrated in FIG. 16.

EXAMPLE 6 Synthesis of benzothiazole-2-carboxamide (8)

Benzothiazole-2-carboxamide was synthesized from ethylbenzothiazole-2-carboxylate according to the following reaction scheme.

In particular, commercially available ethyl benzothiazole-2-carboxylate6 (2.4 mmol, 0.5 g) was dissolved in 34 mL MeOH and purged with NH₃ gas.Conversion to the product was monitored by TLC (3 Hexane: 2 EtOAc).Following complete conversion to the product 8 (˜30 min), the solventwas removed in vacuo. Agilent HPLC displayed 100% conversion to thedesired product 8 with a single peak at ˜8.0 min. The solid whitecompound was used in the subsequent step without purification. ESI-MS:m/z calcd for C₈H₆N₂OS 179.02 (M+H)⁺, 179.88 found (100%) (see LC/MSspectra of FIG. 17A and NMR spectra of FIG. 17B).

EXAMPLE 7 Synthesis of 6-nitrobenzothiazole-2-carboxamide (9)

6-nitrobenzothiazole-2-carboxamide was synthesized frombenzothiazole-2-carboxamide according to the following reaction scheme.

In particular, 2-carboxamide-benzothiazole 8 (0.88 mmol, 0.156 g) wassuspended in 0.655 ml conc. H₂SO₄ at 0° C. 1.3 equivalents ofconcentrated HNO₃ (1.1 mmol, 0.069 g) was added portionwise over 10 minto the stirring solution; not to exceed 0° C. The reaction remained at0° C. for ˜6 h and continued overnight; warming to 25° C. overnight. Thefollowing day the reaction was poured over ice/water forming a whiteprecipitate in solution. The reaction was washed with water, extractedinto EtOAc, and concentrated in vacuo to yield crude product. Attemptsto purify the product were made using gravity silica columnchromatography (Initially 3 Hexane: 7 CH₂Cl₂ followed by 3 Hexane: 2EtOAc) to yield a pure white solid (˜30% yield). (see LC/MS spectra ofFIG. 18). Further NMR can be performed to detect the monofunctionalbenzothiazole comprising the N_(O2) at position C6 versus thiazolesubstituted in the C4, C5, or C7 positions.

EXAMPLE 8 Synthesis of 2-cyanobenzothiazole (10)

2-Cyanobenzothiazole was synthesized from benzothiazole-2-carboxamideaccording to the following reaction scheme.

Benzothiazole-2-carboxamide 8 (0.61 mmol, 0.1 g) was dissolved inanhydrous pyridine (0.080 mol, 6.5 ml) and stirred under N₂ at 0° C. Thetemperature of the reaction was dropped to 0° C. during dropwiseaddition of POCl₃ (0.015 mol, 1.4 ml). The solution immediately changedfrom clear to a pale pink coloring with addition of POCl₃. After 20 min,the acetone/ice bath was removed and the reaction was stirred at RT for2 h further. The tan/brown-colored reaction was then transferred to alarger reaction flask containing 10 ml EtOAc at 0° C. The reaction wasquenched by the dropwise addition of water. The organic product 10 waswashed with water, extracted with EtOAc, and concentrated in vacuo toyield a light yellow/orange product. TLC analysis (3 Hexane: 2 EtOAc)and Agilent HPLC displayed 100% conversion to the desired product(single peak at 10.5 min). The compound was used in the subsequent stepwithout purification (95% yield). ESI-MS: m/z calcd for C₈H₄N₂S 161.01(M +H)⁺, 160.86 found (100%). (see LC/MS spectra of FIG. 19A and NMRspectra of FIG. 19B).

EXAMPLE 9 Synthesis of 2-cyano-6-nitrobenzothiazole (11)

2-Cyano-6-nitrobenzothiazole was synthesized from 2-cyanobenzothiazoleaccording to the following reaction scheme.

In particular 2-cyano-benzothiazole 10 (0.84 mmol, 0.134 g) wassuspended in 0.563 ml conc. H₂SO₄ at 0° C. 1.3 equivalents ofconcentrated HNO₃ (1.1 mmol, 0.069 g) was added portionwise over 10 minto the stirring solution; not to exceed 0° C. over a total of ˜45 min.The reaction proceeded further for 2 h and at 5 h the product wasevident. The reaction was then poured over ice/water, extracted intoEtOAc, and concentrated in vacuo to yield a crude yellow residue 11. Foranalytical characterization, a small portion was purified bysemi-preparative HPLC at 10 mL/min on a Waters preparative machine(Waters Symmetry prep C18, 7 μm, 19×300 mm column, photodiode arraydetection) with a linear gradient from 90% H₂O (0.1% TFA) to 50% MeCN(0.1% TFA) for 30 min yielding a large product peak at ˜27 minutes. Thefractions were collected and lyophilized using Kinetics Flexi-Dryfreeze-dryer.

Further NMR can be performed to detect the monofunctional benzothiazolecomprising the NO₂ at position C6 versus thiazole substituted in the C4,C5, or C7 positions.

EXAMPLE 10 Synthesis of Tyrosine-Aminocyano Derivative of2-cyano-6-aminobenzothiazole

The tyrosine-aminocyano derivative of 2-cyano-6-aminobenzothiazole wassynthesized according to the following reaction scheme.

Tyrosine protected amino acid (0.32 mmoles, 0.11 g) was dissolved in 4mL of anhydrous THF and stirred under N₂ at 0° C. in the dark.N-methylmorpholine (2 eq, 0.64 mmol, 0.070 mL) and isobutylchloroformate (1.3 eq, 0.42 mmol, 0.055 mL) were added dropwise to thereaction at 0° C. and stirred for 30 min in the dark. Separately,purified 2-cyano-6-aminobenzothiazole 5 (0.32 mmol, 0.056 g) wasdissolved in 1 mL of anhydrous THF and then added dropwise to thereaction flask over a period of 30 min. The reaction was stirred underN₂ in the dark at 0° C. for 2 h, followed by an overnight stir at roomtemperature in the dark. 24 h later the anhydrous THF was removed invacuo. The product 12 was dissolved in EtOAc and washed with saturatedNaHCO₃ and NaCl to quench any remaining isobutyl chloroformate. Theorganic layer was collected, dried with MgSO₄, and evaporated todryness. The protected group on the tyrosine portion of the product wasremoved using 20% TFA in anhydrous CH₂Cl₂; stirred at room temperaturein the dark for 2 h. Following deprotection, the product 12 wasevaporated to dryness and placed on vacuum.

The residue was redissolved in 50/50 0.1% TFA in MeCN & H₂O (+drops ofDMF to solubilize) and any solid residue removed by filtration through a0.45 μm filter. Analytical HPLC with a linear gradient from 95% H₂O(0.1% TFA) to 80% MeCN (0.1% TFA) over 15 min yielded a peak at 7.3 min.The material was purified using the semi-preparative HPLC at 10 mL/minon a Waters preparative machine (Waters Symmetry prep C18, 7 μm, 19×300mm column, photodiode array detection) with a linear gradient from 90%H₂O (0.1% TFA) to 50% MeCN (0.1% TFA) for 30 min. Semi-preparative HPLCfractions at 17 min were collected and lyophilized using KineticsFlexi-Dry freeze-dryer. Compound 12 was identified with LC/MSspectroscopic analysis as illustrated in FIG. 20.

This same procedure can be applied for various other amino acids (e.g.the 20 amino acids) as a means to develop new conjugates asbioluminescence probes that may vary in their kinetic activity(pharmacodynamics/pharmacokinetics) in vitro and in vivo

EXAMPLE 11 Synthesis of tyrosine-D-aminoluciferin from AminoAcid-Conjugated 2-cyano-6-aminobenzothiazole Intermediate

The tyrosine-d-aminoluciferin compound was synthesized according to thefollowing reaction scheme

The deprotected and purified amino acid-conjugated2-cyano-6-aminobenzothiazole (e.g. tyrosine conjugated; 0.048 mmoles,0.024 g) was dissolved in anhydrous THF (0.5 mL), followed by dropwiseaddition of D-cysteine (1.2 eq, 0.06 mmoles, 0.011 g, in 0.056 mL;adjusted to pH 8) while stifling the reaction at RT in the dark for 2 h.After 2 h, the reaction was evaporated to dryness and placed on vacuum.The residue was re-dissolved in a mixture of anhy. THF and any solidresidue removed by filtration through a 0.45 μm filter. The solventswere removed in vacuo.

EXAMPLE 12 Synthesis of Peptide Conjugated2-cyano-6-aminoacid-aminobenzothiazole from the Amino Acid-Conjugated2-cyano-6-aminobenzothiazole Intermediate

The peptide-conjugated 2-cyano-6-aminoacid-aminobenzothiazole wassynthesized according to the following reaction scheme.

The peptide sequence was designed to be recognized by a specificprotease in the blood that is indicative of disease (e.g. prostatespecific antigen (PSA), which correlates to prostate cancer; a widevariety of proteases are also present and these levels may change inresponse to biological insults conferred by infection, malignant growthand autoimmune responses¹⁶. The protected peptide sequence (0.033mmoles, 0.067 g) was dissolved in 2.3 mL of anhydrous THF (+small amountof anhydrous DMF), sonicated, and stirred under N₂ at 0° C. in the dark(material in suspension). N-methylmorpholine (2 eq, 0.07 mmol, 0.007 mL)and isobutyl chloroformate (1.3 eq, 0.04 mmol, 0.006 mL) were addeddrop-wise to the reaction at 0° C. and stirred for 30 min in the dark.Separately, purified amino acid-conjugated 2-cyano-6-aminobenzothiazole(e.g. tyrosine conjugated) 12 (0.03 mmol, 0.011 g) was dissolved in 0.2mL of anhydrous THF and then added dropwise to the reaction flask over aperiod of 30 min. The reaction was stirred under N₂ in the dark at 0° C.for 2 h, followed by a 72 h stir at room temperature in the dark. After72 h, the anhydrous THF/DMF was removed in vacuo. The product 14 wasdissolved in EtOAc and washed with saturated NaHCO₃ to quench anyremaining isobutyl chloroformate. The organic layer was collected andevaporated to dryness to yield a bright yellow to golden orange/yellowresidue. The protected group on the peptide portion of the product wasremoved using 50% TFA in anhydrous CH₂Cl₂; stirred at RT in the dark for3 h. Following deprotection, the product 14 was evaporated to drynessand placed on vacuum. Compound 14 was identified with LC/MSspectroscopic analysis as illustrated in FIG. 21.

This same procedure can be applied for various other peptide sequencesas a means to develop new conjugates as bioluminescence probes fordetection of various other proteases that link to disease.

EXAMPLE 13 Synthesis of Peptide Conjugated tyrosine-D-aminoluciferinfrom the Peptide Conjugated2-cyano-6-aminobenzothiazole-tyrosine-Conjugated Intermediate

The peptide-conjugated tyrosine-D-aminoluciferin was synthesizedaccording to the following reaction scheme.

The deprotected and purified peptide-conjugated2-cyano-6-aminobenzothiazole-[amino acid] (0.006 mmoles, 0.008 g) wasdissolved in anhydrous THF (0.01 mL), followed by drop-wise addition ofD-cysteine (1.2 eq, 0.007 mmoles, 0.001 g, 0.001 mL) while stifling thereaction at RT in the dark for 2 h. After 2 h, the reaction wasevaporated to dryness and placed on vacuum. The residue was re-dissolvedin a mixture of anhy. THF and any solid residue removed by filtrationthrough a 0.45 μm filter. The THF was removed in vacuo

EXAMPLE 14 Synthesis of a Carboxylate-Modified Luciferin AminoAcid/Peptide Probe

Chlorotrityl chloride (CTC) resin can be used to synthesize aminoacid/peptide derivatives of D-Luciferin as outlined below. An examplefor phenylalanine is shown below however chlorotryl chloride resin iscommonly used to synthesize peptides, A peptide of any length can besynthesized using standard Fmoc amino add synthesis the D-luciferin canbe conjugated as a last step and the conjugate cleaved from the resin.Purification is using reversed phase high performance liquidchromatography (HPLC)

Reference is made to the reaction Scheme of FIG. 14 showing a schematicillustration of an exemplary reaction scheme.

EXAMPLE 14 Methods Using a Carboxylate-Modified Luciferin AminoAcid/Peptide Probe

Carboxypeptidase Y was used but other carboxypeptidases are expected tobe suitable depending on the nature of the amino acids attached to theD-luciferin as will be understood by a skilled person. CarboxypeptidaseY (sold supported or free in solution) can be used to remove C-terminiamino acids from a peptide. In the case shown here, it will removephenylalanine from the conjugate, releasing the D-luciferin for reactionwith the enzyme. Carboxypeptidase Y is expected to be confirmed asfunctioning optimally using hippuryl-L-phenylalanine, a commerciallyavailable UV-active substrate for carboxypeptidases. Reference is madeto the reaction

The scheme of FIG. 15A showing a schematic illustration of an exemplaryreaction scheme.

A blocking carboxypeptidase activity using a blocking agent (e.g. CONH2)on the C. terminus of the conjugates was investigated as outlined inFIG. 15B.

These determinations are expected to confirm the utility of D-luciferinamino acid I peptide conjugates as extremely sensitive probes forproteolytic action for in vitro and in vivo assays as outlined below.This example shows the C terminus of the substrate blocked as acarboxamide. Only upon proteolytic cleavage of the probe will freecarboxy terminus be revealed for the carboxypeptidase to digest andrelease the D-luciferin for subsequent reaction with luciferase. In thisconnection reference is made to the schematic illustration of FIG. 15C.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the compounds, compositions, systems andmethods of the disclosure, and are not intended to limit the scope ofwhat the inventors regard as their disclosure. All patents andpublications mentioned in the specification are indicative of the levelsof skill of those skilled in the art to which the disclosure pertains.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background, Summary, Detailed Description, andExamples is hereby incorporated herein by reference. All referencescited in this disclosure are incorporated by reference to the sameextent as if each reference had been incorporated by reference in itsentirety individually.

It is to be understood that the disclosures are not limited toparticular compositions or biological systems, which can, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting. As used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. The term “plurality”includes two or more referents unless the content clearly dictatesotherwise. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which the disclosure pertains.

When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and possible subcombinationsof the group are intended to be individually included in the disclosure.Every combination of components or materials described or exemplifiedherein can be used to practice the disclosure, unless otherwise stated.One of ordinary skill in the art will appreciate that methods, deviceelements, and materials other than those specifically exemplified can beemployed in the practice of the disclosure without resort to undueexperimentation. All art-known functional equivalents, of any suchmethods, device elements, and materials are intended to be included inthis disclosure. Whenever a range is given in the specification, forexample, a temperature range, a frequency range, a time range, or acomposition range, all intermediate ranges and all subranges, as wellas, all individual values included in the ranges given are intended tobe included in the disclosure. Any one or more individual members of arange or group disclosed herein can be excluded from a claim of thisdisclosure. The disclosure illustratively described herein suitably canbe practiced in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

A number of embodiments of the disclosure have been described. Thespecific embodiments provided herein are examples of useful embodimentsof the disclosure and it will be apparent to one skilled in the art thatthe disclosure can be carried out using a large number of variations ofthe monofunctional benzothiazoles, compositions, methods steps, andsystems set forth in the present description. As will be obvious to oneof skill in the art, methods and devices useful for the present methodscan include a large number of optional composition and processingelements and steps.

It will be understood that various modifications may be made withoutdeparting from the spirit and scope of the present disclosure.Accordingly, other embodiments are within the scope of the followingclaims.

REFERENCES

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1. A method to provide a peptide conjugated to6-amino-6-deoxy-D-luciferin, the method comprising conjugating an aminoacid with a 2-cyano-6-aminobenzothiazole to provide a2-cyano-6-amino-aminoacid-benzothiazole, the conjugating performed toallow formation of a bond between the amino group of6-amino-6-deoxy-D-luciferin and a carboxylic group of the amino acid;conjugating the peptide with the 2-cyano-6-amino-aminoacid-benzothiazoleto provide a 2-cyano-6-amino-peptide-benzothiazole; and reacting the2-cyano-6-amino-peptide-benzothiazole with a D-cysteine to provide a6-amino-peptide-6-deoxy-D-luciferin, wherein the bond is a peptide bond,carbamate bond or a urea/thiourea bond.
 2. The method of claim 1,wherein the bond is a peptide bond.
 3. The method of claim 1, whereinthe amino acid is an amino acid having a side chain hydrophobic,hydrophilic, acid or basic in nature.
 4. The method of claim 3, whereinthe amino acid is Alanine, Isoleucine, Leucine, Methionine,Phenylalanine, Tryptophan, Tyrosine and Valine.
 5. The method of claim3, wherein the amino acid is Serine, Threonine, Asparagine andGlutamine.
 6. The method of claim 3, wherein the amino acid is Arginine,histidine and Lysine, Aspartic acid and Glutamic acid.
 7. The method ofclaim 1, wherein the peptide comprises at least 5 amino acid residues.8. The method of claim 7, wherein the peptide comprises at least 6 or 7amino acid residues.
 9. The method of claim 1, wherein the peptidefurther comprises a label.
 10. A system to provide a peptide conjugatedto a 6-amino-6-deoxy-D-luciferin, the system comprising one or morepeptides, amino acids, 2-cyano-6-aminobenzothiazole,2-cyano-6-amino-aminoacid-benzothiazoles and/or2-cyano-6-amino-peptide-benzothiazoles for simultaneous combined orsequential use in the method to provide a peptide labeled with6-amino-6-deoxy-D-luciferin according to claim
 1. 11. An intermediate inthe synthesis of an amino acid labeled with 6-amino-6-deoxy-D-luciferin,the intermediate being a 2-cyano-6-aminobenzothiazole of formula (III)

wherein R1 is a single amino acid and R2 is a peptide attached to theremainder of the compound of formula (IV) through a peptide bond acarbamate bond or a urea/thiourea bond.
 12. A labeled peptide obtainableby the method to provide a peptide conjugated to a6-amino-6-deoxy-D-luciferin according to claim 1.